Two homeostatic tasks that are central to the life of a cell are the regulation of intracellular pH (pHi) and the regulation of cell volume (v cell). These problems are interrelated because some transporters (e.g., the Na+/H+ exchanger) that regulate v cell also regulate pHi. The work proposed here would continue research that has been a major focus of the principal investigator over 15 years. The proposal has two Specific Aims. The first Aim focuses on pHi regulation, specifically, a newly discovered K/HCO3 cotransporter that normally moves HCO3 and K+ out of the cell. This work will be performed on internally dialyzed squid giant axons during annual 7-week visits to the Marine Biological Laboratory (Woods Hole, MA). This Aim has 5 parts: (a) Determine the magnitude and properties of the 86Rb efflux that accompanies the HCO3 efflux (the cotransporter moves Rb+ as well as K+); (b) Determine the [K+]i dependence of the HCO3 efflux mediated by the cotransporter. The HCO3 flux is computed from pHi measurements made with microelectrodes. This work exploits the extremely powerful techniques (recently invented in the principal investigator's laboratory) for making out-of-equilibrium (OOE) CO2/HCO3 solutions. The axons will be exposed to a "pure" CO2 solution (normal pH and [CO2], but minimal HCO3), so that the intracellular HCO3 generated by the entering CO2 will generate a large in-to-out HCO3 gradient, maximizing the HCO3 efflux and minimizing influx; (c) Using a similar approach, determine the (HOC3]i dependence of the HCO3 efflux mediated by cotransporter; (d) Identify quaternary amines (QAs) that inhibit the cotransporter with more potency than TEA+. There are no other known inhibitors of the cotransporter. In the previous funding period, the principal investigator found that tetramethyl-, -ethyl-, propyl- and -butylammonium all inhibit the cotransporter from the intracellular side; and (e) Determine if the QAs are competitive with internal K+. The second Aim, focuses on v cell regulation, specifically, the signal-transduction mechanism by which cell shrinkage activates Na+/H+ exchange. This work will be performed at Yale on Xenopus oocytes, and has five parts: (a) Determine if low pHi accelerates the signal-transduction process; (b) Determine if hypertonicity can activate the Na+/H+ exchanger before there is a macroscopic shrinkage; (c) Identify the heterotrimeric G protein responsible for mediating shrinkage-induced activation of Na+/H+ exchange; (d) Test the macromolecular-crowding hypothesis, which suggests that increased concentrations of large molecules is part of the shrinkage signal; and (e) Determine if shrinkage activates mammalian Na-H exchanger expressed in oocytes. The proposed work on squid axons will greatly increase our understanding of the K/HCO3 cotransporter, which may be the major transporter that lowers pHi in mammalian neurons and astrocytes. The oocyte work will demonstrate whether the shrinkage sensor is at the plasma membrane, and identify the G-protein alpha subunit. The oocyte work also will elucidate the role played by pHi in controlling the rate at which shrinkage activates the Na+/H+ exchanger.